Method for detecting L-pyroglutamic acid and methionine sulfoxide in compound amino acid injection

Abstract

The invention discloses a method for detecting L-pyroglutamic acid and methionine sulfoxide in a compound amino acid injection, and relates to the technical field of medicine detection.Chromatographic conditions of a high performance liquid chromatography are as follows: gradient elution is performed by adopting a chromatographic column of an octadecylsilane chemically bonded silica filler and a mobile phase consisting of a mobile phase A and a mobile phase B, a detector is an ultraviolet detector, the detection wavelength is 205-210 nm, the flow velocity of a mobile phase is 0.7-1.2 ml / min, the sample size is 5-25 [mu] l, the column temperature is 20-25 DEG C, the mobile phase A is a mixed solution of an ion pair reagent, a phosphate buffer solution, methanol and acetonitrile, and the mobile phase B is one or more of a methanol solution, an acetonitrile solution and a phosphate buffer solution; the detection process comprises the following steps: injecting a test solution into a liquid chromatograph, recording a chromatogram, and calculating the contents of methionine sulfoxide and L-pyroglutamic acid by peak areas according to an external standard method.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting L-pyroglutamic acid and methionine sulfoxide in a compound amino acid injection. Background Art

[0002] Compound amino acid injection mainly contains a variety of amino acids and other raw materials and auxiliary materials. It is made by high-temperature steam sterilization and nitrogen filling. It is a parenteral nutrition solution with wide clinical application. Methionine and glutamic acid are more common components of compound amino acid injection, but methionine is an easily oxidized amino acid, and its oxidative degradation mainly produces methionine sulfoxide, while glutamic acid is easily condensed under high temperature conditions to form L-pyroglutamic acid, which causes the compound amino acid injection to deteriorate. In practical applications, we detect the content of methionine sulfoxide in compound amino acid injection to understand the effect of oxygen in the environment on the oxidative degradation state of compound amino acid injection; and detect the content of L-pyroglutamic acid to understand the influence of high-temperature sterilization procedures and storage environment on the stability of compound amino acid injection during the production process of the corresponding batch of drugs.

[0003] At present, high performance liquid chromatography (HPLC) is usually used to determine the impurities in compound amino acid injection. For example: Shao Tianshu et al. established a method for simultaneous determination of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA-Ⅱ) by pre-column derivatization-HPLC; Li Li et al. used HPLC to directly determine methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA); Jiang Ge et al. used HPLC and HILIC columns to determine the related substances of methionine in pediatric compound amino acid injection, and found that the main degradation impurity of methionine was methionine sulfoxide; Zhu Yijuan et al. used pre-column derivatization high performance liquid phase fluorescence detection to determine the content of methionine sulfoxide in compound amino acid injection; Shan Guangzhi et al. used high performance liquid chromatography to establish a compound amino acid injection. Detection method of pyroglutamic acid content in compound amino acid injection; Xie Shenggu et al. established an HPLC method for determining the content of pyroglutamic acid in compound amino acid injection (18AA); Su Zhe et al. established an HPLC method for determining the content of pyroglutamic acid and acetate in compound amino acid injection (20AA); Wang Ji et al. established an HPLC method for determining the contents of three impurities including pyroglutamic acid, cyclic (glycyl-glutamine) and cyclic (glycyl-tyrosine) in compound amino acid (15) dipeptide (2) injection; Li Li et al. established a limited detection method for the determination of the impurity pyroglutamic acid in compound amino acid injection (18AA), (18AA-I), (18AA-II) and (18AA-V) by high performance liquid chromatography (HPLC).

[0004] It is not difficult to see that the current determination of methionine sulfoxide and L-pyroglutamic acid in compound amino acid injection requires two different analytical methods, which are inefficient and not conducive to the overall quality control of compound amino acid injection. Some analytical methods have harsh conditions. For example, Li Li et al. used HPLC to directly determine methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA) at the same time. The column temperature was 4°C, and the column oven required an additional cooling kit, which was difficult to achieve under general laboratory conditions; Jiang Ge et al. used HPLC to determine the related substances of methionine in pediatric compound amino acid injection, using HILIC columns. This type of column has harsh conditions, long equilibrium time, and the price of the column itself is high; Shao Tianshu, Zhu Yijuan et al. all used pre-column derivatization fluorescence chromatography to determine methionine sulfoxide, which is cumbersome. It can be seen that establishing a method to simultaneously analyze methionine sulfoxide and L-pyroglutamic acid can reduce resource waste and improve inspection efficiency. Summary of the invention

[0005] In view of the above problems in the prior art, the present invention provides a method for detecting L-pyroglutamic acid and methionine sulfoxide in a compound amino acid injection, which can directly, conveniently and quickly detect methionine sulfoxide and pyroglutamic acid in the compound amino acid injection qualitatively or quantitatively under one chromatographic condition, so as to solve the technical problems of low inspection efficiency and complicated operation in the prior art for simultaneously detecting the presence of L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The invention discloses a method for detecting L-pyroglutamic acid and methionine sulfoxide in a compound amino acid injection. The method adopts high performance liquid chromatography to simultaneously detect L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection. The method is characterized in that the chromatographic conditions include: using a chromatographic column with an octadecylsilane bonded silica gel filler, performing gradient elution with a mobile phase composed of a mobile phase A and a mobile phase B, a detector being an ultraviolet detector, a detection wavelength of 205 to 210 nm, a mobile phase flow rate of 0.7 ml / min to 1.2 ml / min, an injection volume of 5 to 25 μl, and a column temperature of 20 to 25° C., wherein the mobile phase A is a mixed solution of an ion pair reagent, a phosphate buffer solution, methanol, and acetonitrile, and the mobile phase B is one or more of a methanol solution, an acetonitrile solution, and a phosphate buffer solution; and the detection process comprises: injecting a test sample solution into a liquid chromatograph, recording a chromatogram, and calculating the contents of methionine sulfoxide and L-pyroglutamic acid by peak area according to an external standard method.

[0008] Preferably, the phosphate buffer solution is a solution with a concentration of 0.005 mol / L to 0.020 mol / L, and in the mobile phase A, the volume ratio of the phosphate buffer solution to methanol and acetonitrile is 1000:(10-20):(20-10), and the mass percentage of the ion pair reagent in the phosphate buffer solution is 0.2% to 0.5%.

[0009] Furthermore, the phosphate in the phosphate buffer solution is one or both of ammonium dihydrogen phosphate and diammonium hydrogen phosphate, and the ion pair reagent is sodium heptane sulfonate or sodium octane sulfonate.

[0010] Furthermore, when preparing mobile phase A, the ion pair reagent is first added to the phosphate buffer solution, and then the pH value of the phosphate buffer solution containing the ion pair reagent is adjusted to 1.6-2.3 using phosphoric acid, and then the phosphate buffer solution containing the ion pair reagent is mixed with methanol and acetonitrile to obtain mobile phase A.

[0011] Preferably, the phosphate buffer solution is a solution with a concentration of 0.005 mol / L to 0.020 mol / L, and in the mobile phase B, the volume percentage of the phosphate buffer solution is 0 to 20%, the volume percentage of methanol is 0 to 100%, and the volume percentage of acetonitrile is 0 to 100%.

[0012] Preferably, the test sample is diluted 10 times using mobile phase A before detection.

[0013] Preferably, the compound amino acid injection is one of compound amino acid injection (18AA), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II), compound amino acid injection (18AA-III), compound amino acid injection (18AA-IV), and compound amino acid injection (18AA-V).

[0014] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] 1. The method for detecting methionine sulfoxide and L-pyroglutamic acid impurities provided by the present invention can simultaneously analyze methionine sulfoxide and L-glutamic acid impurities in the compound amino acid injection. The method has strong specificity, high sensitivity and accurate detection results. The content of methionine sulfoxide and L-pyroglutamic acid in the compound amino acid injection can be accurately evaluated, and the storage state and production quality of the compound amino acid injection can be further determined.

[0016] 2. The present invention adopts ultraviolet detector for direct measurement, avoiding the use of derivatization reagents, and has the advantages of being environmentally friendly and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the present invention and the technical solutions in the prior art, the following briefly introduces the drawings required in the description of the present invention and the prior art. The drawings in the following description are only some implementation cases of the present invention, among which:

[0018] Figure 1 It is the blank solvent (mobile phase A) chromatogram in Example 1;

[0019] Figure 2 is the blank matrix chromatogram in Example 1;

[0020] Figure 3 The chromatogram of the reference substance solution in Example 1 (L-pyroglutamic acid: 5.013 min, methionine sulfoxide: 8.834 min);

[0021] Figure 4 This is the chromatogram of the spiked solution in Example 1 (L-pyroglutamic acid: 5.015 min, methionine sulfoxide: 8.828 min);

[0022] Figure 5 is the standard linear relationship diagram of L-pyroglutamic acid, in which the horizontal axis is the concentration (μg / ml) and the vertical axis is the peak area;

[0023] Figure 6 This is a standard linear relationship diagram of methionine sulfoxide, where the horizontal axis is concentration (μg / ml) and the vertical axis is peak area. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and various embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments.

[0025] The present invention uses an Agilent 1260 high performance liquid chromatograph or a Waters e2695 high performance liquid chromatograph, uses a CAPCELL PAK C18 AQ chromatographic column (4.6×250 mm, 5 μm) or a Yuexu Ultimate AQ C18 chromatographic column (4.6×250 mm, 5 μm), detects a reference substance that is a commercially available high-purity reagent, and uses a commercially available chromatographic-grade reagent.

[0026] Example 1

[0027] In this example, L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection were specifically determined, and the compound amino acid injection tested was the compound amino acid injection (18AA-I).

[0028] 1. Solution preparation

[0029] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0030] Mobile phase B: methanol.

[0031] Blank matrix: Prepared according to the concentration of the compound amino acid injection prescription (excluding glutamic acid and methionine).

[0032] Blank matrix solution: Accurately measure 1 ml of blank matrix, place in a 10 ml volumetric flask, and dilute to the mark with mobile phase A.

[0033] Stock solution of reference substance: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances respectively, weigh accurately, add mobile phase A to dissolve them respectively and dilute them quantitatively to prepare a solution containing 500 μg / ml of L-pyroglutamic acid and a solution containing 100 μg / ml of methionine sulfoxide.

[0034] Reference solution: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference stock solutions respectively, accurately weigh and place in a volumetric flask, add mobile phase A to dissolve and quantitatively dilute to prepare a solution containing 50 μg / ml of L-pyroglutamic acid and 10 μg / ml of methionine sulfoxide.

[0035] Spiked solution: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances, weigh accurately, dissolve in blank matrix solution and quantitatively dilute with mobile phase A to prepare a solution containing 50 μg / ml of L-pyroglutamic acid and 10 μg / ml of methionine sulfoxide.

[0036] 2. Detection

[0037] The gradient elution conditions were as follows:

[0038] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0039] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0040] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0041] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0042] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0043] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0044] Column temperature: 25°C

[0045] Detection wavelength: 205nm

[0046] Flow rate: 1ml / min

[0047] Injection volume: 10 μl

[0048] Detection method: Precisely measure blank solvent (mobile phase A), blank matrix solution, reference solution, and spiked solution, and inject them into a high performance liquid chromatograph. The chromatogram is as follows: Figure 1 to Figure 4 The blank solvent chromatogram was used as a control, and the analysis results are shown in Table 1.

[0049] Table 1 Specificity determination results

[0050] Sample name Pyroglutamic acid retention time / min Methionine sulfoxide retention time / min Blank matrix solution / / Reference solution 5.013 8.834 Spike solution 5.011 8.768

[0051] Example 2

[0052] In this example, the quantitative limit and the detection limit were determined for L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection. The compound amino acid injection tested was the compound amino acid injection (18AA-I).

[0053] 1. Solution preparation

[0054] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0055] Mobile phase B: methanol.

[0056] Blank matrix: Prepared according to the concentration of the compound amino acid injection prescription (excluding glutamic acid and methionine).

[0057] Blank matrix solution: Accurately measure 1 ml of blank matrix, place in a 10 ml volumetric flask, and dilute to the mark with mobile phase A.

[0058] Stock solution of reference substance: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances respectively, weigh accurately, add mobile phase A to dissolve them respectively and dilute them quantitatively to prepare a solution containing 5000 μg / ml of L-pyroglutamic acid and a solution containing 1000 μg / ml of methionine sulfoxide.

[0059] Quantitation limit solution: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference stock solutions respectively, place them in volumetric flasks and dilute them step by step with mobile phase A until the signal-to-noise ratio of the chromatographic peaks of L-pyroglutamic acid and methionine sulfoxide is about 10:1, which is used as the quantitation limit solution.

[0060] Detection limit solution: Accurately take an appropriate amount of the quantitative limit solution, place it in a volumetric flask and dilute it with mobile phase A so that the signal-to-noise ratio of the chromatographic peaks of L-pyroglutamic acid and methionine sulfoxide is about 3:1, which is used as the detection limit solution.

[0061] 2. Detection

[0062] The gradient elution conditions were as follows:

[0063] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0064] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0065] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0066] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0067] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0068] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0069] Column temperature: 25°C

[0070] Detection wavelength: 205nm

[0071] Flow rate: 1ml / min

[0072] Injection volume: 10 μl

[0073] Detection method: Accurately measure the quantitative limit solution and the detection limit solution respectively, and inject them into the high performance liquid chromatograph. The results are shown in Table 2.

[0074] Table 2 Results of limit of quantitation and limit of detection

[0075]

[0076]

[0077] Example 3

[0078] In this example, the detection linearity and linear range of L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection were determined. The compound amino acid injection detected was the compound amino acid injection (18AA-I).

[0079] 1. Solution preparation

[0080] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0081] Mobile phase B: methanol.

[0082] Blank matrix: Prepared according to the concentration of the compound amino acid injection prescription (excluding glutamic acid and methionine).

[0083] Blank matrix solution: Accurately measure 1 ml of blank matrix, place in a 10 ml volumetric flask, and dilute to the mark with mobile phase A.

[0084] Stock solution of reference substance: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances respectively, weigh accurately, add mobile phase A to dissolve them respectively and dilute them quantitatively to prepare a solution containing 5000 μg / ml of L-pyroglutamic acid and a solution containing 1000 μg / ml of methionine sulfoxide.

[0085] Quantitation limit solution: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference stock solutions respectively, place them in volumetric flasks and dilute them step by step with mobile phase A until the signal-to-noise ratio of the chromatographic peaks of L-pyroglutamic acid and methionine sulfoxide is about 10:1, which is used as the quantitation limit solution.

[0086] Linear working solution L1: Take 1 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively, place them in a 50 ml volumetric flask, and quantitatively dilute to the scale with mobile phase A to prepare a solution containing 100 μg / ml L-pyroglutamic acid and 20 μg / ml methionine sulfoxide.

[0087] Linear working solution L2: Take 0.75 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively, place them in a 50 ml volumetric flask, and quantitatively dilute to the scale with mobile phase A to prepare a solution containing 75 μg / ml of L-pyroglutamic acid and 15 μg / ml of methionine sulfoxide.

[0088] Linear working solution L3: Take 0.5 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively, place them in a 50 ml volumetric flask, and quantitatively dilute to the scale with mobile phase A to prepare a solution containing 50 μg / ml of L-pyroglutamic acid and 10 μg / ml of methionine sulfoxide.

[0089] Linear working solution L4: Take 0.25 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively, place them in a 50 ml volumetric flask, and quantitatively dilute to the scale with mobile phase A to prepare a solution containing 25 μg / ml of L-pyroglutamic acid and 5 μg / ml of methionine sulfoxide.

[0090] Linear working solution L5: Take 0.1 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively, place them in a 100 ml volumetric flask, and quantitatively dilute to the scale with mobile phase A to prepare a solution containing 5 μg / ml of L-pyroglutamic acid and 1 μg / ml of methionine sulfoxide.

[0091] 2. Detection

[0092] The gradient elution conditions were as follows:

[0093] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0094] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0095] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0096] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0097] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0098] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0099] Column temperature: 25°C

[0100] Detection wavelength: 205nm

[0101] Flow rate: 1ml / min

[0102] Injection volume: 10 μl

[0103] Detection method: Accurately measure the linear working solutions L1 to L5 and the quantitative limit solution, inject them into the high performance liquid chromatograph, and fit the linear equation with the peak area as the Y axis and the concentration as the X axis. The linear relationship results are shown in Figure 5 , Figure 6 and Table 3.

[0104] Table 3 Linear determination results

[0105] Linear equations Range (μg / ml) Correlation coefficient L-Pyroglutamic acid Y=7.1908X-0.4335 0.176～100.346 0.9999 Methionine sulfoxide Y=3.8757X+0.1745 0.297～19.825 0.9999

[0106] Example 4

[0107] In this example, the detection accuracy of L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection was measured, and the compound amino acid injection measured was the compound amino acid injection (18AA-I).

[0108] 1. Solution preparation

[0109] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0110] Mobile phase B: methanol.

[0111] Blank matrix: Prepared according to the concentration of the compound amino acid injection prescription (excluding glutamic acid and methionine).

[0112] Blank matrix solution: Accurately measure 1 ml of blank matrix, place in a 10 ml volumetric flask, and dilute to the mark with mobile phase A.

[0113] Stock solution of reference substance: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances respectively, weigh accurately, add mobile phase A to dissolve them respectively and dilute them quantitatively to prepare a solution containing 5000 μg / ml of L-pyroglutamic acid and a solution containing 1000 μg / ml of methionine sulfoxide.

[0114] Blank matrix spike solution: Accurately measure 1 ml of blank matrix and place it in a 10 ml volumetric flask. Add 0.25 ml, 0.50 ml and 0.75 ml of L-pyroglutamic acid reference stock solution and methionine sulfoxide reference stock solution respectively. Dilute to the scale with mobile phase A. Prepare 3 copies of each concentration in parallel as low, medium and high concentration spike solutions.

[0115] 2. Detection

[0116] The gradient elution conditions were as follows:

[0117] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0118] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0119] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0120] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0121] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0122] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0123] Column temperature: 25°C

[0124] Detection wavelength: 205nm

[0125] Flow rate: 1ml / min

[0126] Injection volume: 10 μl

[0127] Detection method: Precisely measure the blank matrix solution and the blank matrix spiked solution respectively, inject them into the high performance liquid chromatograph, and calculate the spiked recovery rate by the linear equation under Example 3. The results are shown in Tables 4 and 5.

[0128] Table 4 L-pyroglutamic acid spike recovery

[0129]

[0130] Table 5 Methionine sulfoxide spike recovery

[0131]

[0132]

[0133] Example 5

[0134] In this example, the repeatability of L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection was measured, and the compound amino acid injection measured was the compound amino acid injection (18AA-I).

[0135] 1. Solution preparation

[0136] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0137] Mobile phase B: methanol.

[0138] Blank matrix: Prepared according to the concentration of the compound amino acid injection prescription (excluding glutamic acid and methionine).

[0139] Blank matrix solution: Accurately measure 1 ml of blank matrix, place in a 10 ml volumetric flask, and dilute to the mark with mobile phase A.

[0140] Stock solution of reference substance: Take appropriate amount of L-pyroglutamic acid and methionine sulfoxide reference substances respectively, weigh accurately, add mobile phase A to dissolve them respectively and dilute them quantitatively to prepare a solution containing 5000 μg / ml of L-pyroglutamic acid and a solution containing 1000 μg / ml of methionine sulfoxide.

[0141] Repeatability test solution: Accurately measure 1 ml of blank matrix and place it in a 10 ml volumetric flask. Add 0.50 ml of L-pyroglutamic acid reference stock solution and 0.50 ml of methionine sulfoxide reference stock solution respectively. Dilute to the scale with mobile phase A to make a solution containing 50 μg / ml of L-pyroglutamic acid and 10 μg / ml of methionine sulfoxide. Prepare 6 copies in parallel as the repeatability test solution.

[0142] 2. Detection

[0143] The gradient elution conditions were as follows:

[0144] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0145] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0146] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0147] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0148] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0149] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0150] Column temperature: 25°C

[0151] Detection wavelength: 205nm

[0152] Flow rate: 1ml / min

[0153] Injection volume: 10 μl

[0154] Detection method: The repeatability test solution was accurately measured and injected into the high performance liquid chromatograph. The spike recovery rate was calculated using the linear equation under Example 3. The results are shown in Table 6.

[0155] Table 6 Repeatability test results

[0156]

[0157]

[0158] Example 6

[0159] This example actually detects L-pyroglutamic acid and methionine sulfoxide in different batches of compound amino acid injection (18AA-I).

[0160] 1. Solution preparation

[0161] Mobile phase A: Take 1.15 g of ammonium dihydrogen phosphate and 2.7 g of sodium heptane sulfonate, dissolve them in water and dilute to 1000 ml, adjust the pH value to 2.0 with phosphoric acid, and then mix with acetonitrile and methanol in a volume ratio of 1000:13:10 to obtain mobile phase A.

[0162] Mobile phase B: methanol.

[0163] Test solution: Accurately measure 1 ml of compound amino acid injection (18AA-I), place in a 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well.

[0164] 2. Detection

[0165] The gradient elution conditions were as follows:

[0166] Time: 0 min, 100% mobile phase A, 0% mobile phase B

[0167] Time: 15 min, 100% mobile phase A, 0% mobile phase B

[0168] Time: 20 min, 40% mobile phase A, 60% mobile phase B

[0169] Time: 30 min, 40% mobile phase A, 60% mobile phase B

[0170] Time: 35 min, 100% mobile phase A, 0% mobile phase B

[0171] Time: 60 min, 100% mobile phase A, 0% mobile phase B

[0172] Column temperature: 25°C

[0173] Detection wavelength: 205nm

[0174] Flow rate: 1ml / min

[0175] Injection volume: 10 μl

[0176] Detection method: Accurately measure different batches of test sample solutions, inject them into high performance liquid chromatography, and calculate the contents of L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection (18AA-I) sample using the linear equation under Example 3. The results are shown in Table 7.

[0177] Table 7 L-pyroglutamic acid and methionine sulfoxide determination results (μg / ml)

[0178] Test sample 1 Test sample 2 Test sample 3 Test sample 4 Test sample 5 Test sample 6 L-Pyroglutamic acid 51.983 53.127 49.767 34.183 48.910 49.994 Methionine sulfoxide 0.297 0.305 0.350 0.406 0.316 0.336

[0179] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for detecting L-pyroglutamic acid and methionine sulfoxide in a compound amino acid injection, using high performance liquid chromatography to simultaneously detect L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection, characterized in that: The chromatographic conditions include: using a chromatographic column with octadecylsilane bonded silica gel filler, gradient elution with a mobile phase composed of mobile phase A and mobile phase B, a UV detector, a detection wavelength of 205-210 nm, a mobile phase flow rate of 0.7 ml / min-1.2 ml / min, an injection volume of 5-25 μl, and a column temperature of 20-25° C., wherein the mobile phase A is a mixed solution of an ion pair reagent, a phosphate buffer solution, methanol, and acetonitrile, and the mobile phase B is one or more of a methanol solution, an acetonitrile solution, and a phosphate buffer solution; the detection process is: taking a test solution and injecting it into a liquid chromatograph, recording a chromatogram, and calculating the contents of methionine sulfoxide and L-pyroglutamic acid by peak area according to an external standard method.

2. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 1, characterized in that: The phosphate buffer solution is a solution with a concentration of 0.005 mol / L to 0.020 mol / L. In the mobile phase A, the volume ratio of the phosphate buffer solution to methanol and acetonitrile is 1000:(10-20):(20-10), and the mass percentage of the ion pair reagent in the phosphate buffer solution is 0.2% to 0.5%.

3. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 2, characterized in that: The phosphate in the phosphate buffer solution is one or both of ammonium dihydrogen phosphate and diammonium hydrogen phosphate, and the ion pair reagent is sodium heptane sulfonate or sodium octane sulfonate.

4. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 2, characterized in that: When preparing mobile phase A, first add the ion pair reagent into the phosphate buffer solution, then use phosphoric acid to adjust the pH value of the phosphate buffer solution containing the ion pair reagent to 1.6-2.3, and then mix the phosphate buffer solution containing the ion pair reagent with methanol and acetonitrile to obtain mobile phase A.

5. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 1, characterized in that: The phosphate buffer solution is a solution with a concentration of 0.005mol / L to 0.020mol / L. In the mobile phase B, the volume percentage of the phosphate buffer solution is 0 to 20%, the volume percentage of methanol is 0 to 100%, and the volume percentage of acetonitrile is 0 to 100%.

6. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 1, characterized in that: Before detection, dilute the test sample 10 times using mobile phase A.

7. The method for detecting L-pyroglutamic acid and methionine sulfoxide in the compound amino acid injection according to claim 1, characterized in that: The compound amino acid injection is one of compound amino acid injection (18AA), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II), compound amino acid injection (18AA-III), compound amino acid injection (18AA-IV), and compound amino acid injection (18AA-V).

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